Comparison of the effects of feeding Indian fish liver oils supplemented with or without cholesterol and bile salts on certain enzymes in liver, heart and serum of ratsKG Tanksale, NG Magar
Department of Biochemistry and Nutrition, Seth G. S. Medical College, Parel, Bombay-400 012., India
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 731609
Source of Support: None, Conflict of Interest: None
The enzymes viz. glucose-6-P-dehydrogenase (EC 184.108.40.206), cholesterol esterase (EC 220.127.116.11), aspartate amino transferase (EC 18.104.22.168) and alanine amino transferase (22.214.171.124) are intimately related to lipid metabolism. Hence their activities are bound to be affected by the type of dietary fat and substances like bile salts and cholesterol which also influence the lipid metabolism. This relationship between dietary lipid constituent and enzymes was studied in albino rats maintained on diets containing three Indian Shark Liver Oils viz. Waghbeer, Khada mushi and Pisori supplemented with or without cholesterol and bile salts, Enzyme activities were studied in liver, heart and serum. It was noted that higher unsaturation of dietary fat increased the activity of glucose-6-Pdehydrogenase enzyme while activities of transaminases and cholesterol esterase were lowered. Addition of cholesterol and bile salts to these diets decreased the activity of glucose-6-P-dehydrogenase and hydrolytic activity of cholesterol esterase. There was increase in the activities of transaminases and esterifying activity of cholesterol esterase due to supplementation with cholesterol and bile salts.
It has been shown in rats that dietary proteins, fats and carbohydrates markedly affect various enzyme systems, ,,, particularly those related to lipid metabolism. Perez et al  have also shown that glucose-6-phosphate dehydrogenase (EC 126.96.36.199) activity increases with high levels of proteins and decreases with high fat diet.  Effect of fat free diet and Essential Fatty Acid (EFA) deficient diet on cholesterol esterase (EC 188.8.131.52) activity has been studied. , Influence of cholesterol and bile salt feeding on certain enzymes related to lipid metabolism has been studied by several authors. ,, Century and Horwit  have studied the influence of Poly Unsaturated Fatty Acids (PUFA) on certain enzymes. These results, however, are inconclusive and hence the present studies were undertaken. In this study we used three Indian Fish Liver Oils viz. Waghbeer (Galeocerdo tigrinus), Khada mushi (Carcharinus melanopterus) and Pisori (Carcharinus limbatus) which have been reported  to have low PUFA activity. These fish liver oils are the major source for commercial extraction of vitamin A  and certain aspects of their nutritive value have beer reported. ,
Analysis of Shark Liver oils: The three fish liver oils were obtained from Fisheries Technological Laboratories Bombay. Chemical constants like acid value, saponification value, iodine value peroxide value and unsaponifiable matter were determined by standard methods of A.O.C.S. 
Animal Experiment: Weanling mile .and female albino , rats were used for the present investigation. These rats were divided into five groups each having six males (sub-group A) and six females (sub-group B). All the animals were housed in individual cages throughout the experimental period. They were fed with diets containing Hegsted's modified salt mixture  and Schultz vitamin mixture  as shown in [Table 1]. Fresh diets and water were given on ad-libitum basis for a period of eight weeks. Daily food consumption was noted to see whether it was normal and consistent.
After the experimental period rats were fasted overnight and were sacrificed and blood was collected by cardiac puncture. Liver and heart tissues were excised quickly, blotted and weighed quantities were taken for analyses. Tissue homogenates were prepared in 0.25 M ice cold sucrose solution. Proteins were estimated by the method of Gornall  , Glucose-6 phosphate dehydrogenase (EC 184.108.40.206) by the method of Kornberg and Horecker,  cholesterol esterase (EC 220.127.116.11) by the method of Murthy and Ganguly  and aspartate amino transferase (AS-At) (EC 18.104.22.168) and alanine amino transferase (Al-At) (EC 22.214.171.124) by the method of Reitm..aand Frankel. 
[Table 2] shows chemical constants of the three Shark Liver oils. Khada mushi has the highest iodine value and that of Waghbeer oil is the lowest.
[Table 3] and [Table 4] show the data of enzyme activities from liver, heart and serum of rats in various groups. There is no statistical dfference in the results on the basis of sex difference of the animals in any of the groups studied.
Activity of glucose-6-P-dehydrogenase was found to be significantly higher in liver, heart and serum of rats fed diets containing Khada mushi oil as compared to that of rats fed diets containing Waghbeer oil. When cholesterol was added to diets containing Waghbeer oil there was significant suppression of activity of this enzyme both in males and females. Further addition of bile salts to the diet resulted in further and significant decrease in the activity of glucose-6-P-dehydrogenase.
Cholesterol esterase enzyme was studied for its hydrolytic as well as esterifying activity. The activities were significantly higher in serum of rats fed diets containing Waghbeer oil. On addition of cholesterol to the diet containing Waghbeer oil, the hydrolytic activity was significantly lowered while esterfying activity was increased significantly. Additional supplementation with bile salts caused further decrease in the hydrolylic activity and increase in the esterifying activity. These changes were statistically significant.
Activities of both aspartate and alanine amino transferases were significantly higher in the liver, heart and serum of rats in Group 1 as compared to Group 2. When the diet containing Waghbeer oil was supplemented with cholesterol and bile salts, there was a significant rise in the activities of these enzymes in the tissues mentioned above. In case of rats fed Pisori oil, no significant differences were observed in the activity of any of the enzymes studied in various tissues and sera as compared to those observed in rats fed Waghbeer oil.
The enzyme activities did not show any difference on the basis of sex difference of the animals. Differences, however, were observed due to inclusion of three shark liver oils characterised by different iodine values, absence of linoleic acid and low PUFA content  in the diet.
The relation between the activity of glucose-6-P-dehydrogenase and unsaturation or PUFA content of dietary lipid was shown by Tepperman and Tepperman  as a feed back mechanism. The activity of this enzyme was further shown to be related to lipogenesis in the liver. , The activity became less with decreased lipogenesis. Waghbeer oil feeding resulted into decreased activity of glucose-6-Pdehydrogenase in the liver, heart and serum where hepatic activity was related to lipogenesis. There was a significant difference between the activities of glucose-6-P-dehydrogenase shown by rats fed Waghbeer oil and that shown by rats fed Khada mushi oil. This was possibly due to difference in the iodine values and fatty acid composition of these two fish liver oils. 
Effect of feeding cholesterol showed further significant decrease in the activity of glucose-6-P-dehydrogenase. According to Tsai and Dyer  feeding cholesterol resulted into its accumulation which was a probable cause of the decreased activity of glucose-6-P-dehydrogenase. These authors  have also shown that feeding cholesterol and bile acids lowered the activity of glucose-6-P-dehydrogenase still further due to enhanced accumulation of cholesterol. Observations in the present investigation also confirm the above findings.
Activities of amino transferases are connected with pyruvate and oxalo-acetate metabolism which in turn is related to lipid metabolism through gluconeogenesis or through lactate/pyruvate ratio. According to Yeh et al  dietary lipid alters lactate/pyruvate ratio and ultimately NADH/NAD ratio which serves as an index of availability of reducing equivalents needed for lipogenesis. In the present study feeding fish liver oil with higher unsaturation viz. Khada mushi oil, caused significant decrease in the activities of these enzymes as compared to the data obtained in rats fed Waghbeer oil. Addition of cholesterol and bile salts to the diet containing Waghbeer oil increased the activity of these enzymes and increased lipogenesis may be the probable cause.
Conversion of cholesterol esters into free cholesterol and re-esterification is due to the activity of cholesterol esterase and related to the transport of cholesterol.  Dietary fat influences the esterifying and hydrolytic activity of cholesterol esterase.  In the present investigation three fish liver oils of different iodine values viz. 100 (Waghbeer oil), 143 (Khada mushi oil) and 114 (Pisori oil) were used. As the unsaturation of dietary fat increased there was decrease in the esterifying activity with corresponding decrease in hydrolytic activity of cholesterol esterase and these findings are in agreement with those of Goodman,  Nestle and Monger,  and Glomset  , It was further shown by Takeuchi and Yamamura  that addition of cholesterol to the diet increased the esterifying activity. In the present study with addition of cholesterol to the diet containing Waghbeer oil there was a significant increase in the esterifying activity and decrease in hydrolytic activity and further supplementation with bile salts increased the esterifying activity still further and decreased the hydrolytic activity also. According to Trze ciak and Boyed  cAMP levels in plasm, are related to cholesterol esterase. It is therefore, probable that unsaturation: dietary fat, cholesterol and bile salt influence cholesterol esterase through this mechanism involving cAMP.
Authors are thankful to the Dean, Seth G. S. Medical College, Bombay-400 012 for the permission to publish this article and to the Assistant Director, Fisheries Technological Laboratories, Bombay for supplying the fish liver oils.
[Table 1], [Table 2], [Table 3], [Table 4]